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Mitochondrial damage activates the NLRP10 inflammasome

Nature Immunology volume 24, pages 595–603 (2023)Cite this article

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Abstract

Upon detecting pathogens or cell stress, several NOD-like receptors (NLRs) form inflammasome complexes with the adapter ASC and caspase-1, inducing gasdermin D (GSDMD)-dependent cell death and maturation and release of IL-1β and IL-18. The triggers and activation mechanisms of several inflammasome-forming sensors are not well understood. Here we show that mitochondrial damage activates the NLRP10 inflammasome, leading to ASC speck formation and caspase-1-dependent cytokine release. While the AIM2 inflammasome can also sense mitochondrial demise by detecting mitochondrial DNA (mtDNA) in the cytosol, NLRP10 monitors mitochondrial integrity in an mtDNA-independent manner, suggesting the recognition of distinct molecular entities displayed by the damaged organelles. NLRP10 is highly expressed in differentiated human keratinocytes, in which it can also assemble an inflammasome. Our study shows that this inflammasome surveils mitochondrial integrity. These findings might also lead to a better understanding of mitochondria-linked inflammatory diseases.

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Fig. 1: m-3M3FBS triggers mitochondrial damage and NLRP10 inflammasome activation.
Fig. 2: Mitochondrial permeability transition activates NLRP10 and AIM2 inflammasomes.
Fig. 3: Molecular characterization of NLRP10 activation.
Fig. 4: Keratinocyte differentiation drives NLRP10 expression, enabling responses to mitochondrial damage.

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Data availability

Representative images for all ASC speck formation quantifications are deposited in Mendeley Data using the following link: https://data.mendeley.com/datasets/42fsz64kn5/1. Source data are provided with this paper. All other data are available in the article Supplementary files or from the corresponding author upon reasonable request.

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Acknowledgements

We thank H. Beckert, G. L. Horvath, L. Maus and P. Stausberg from the Microscopy Core Facility of the Medical Faculty at the University of Bonn for providing help, services and devices funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)–Projektnummer 13123509, 388171357 and 169331223. This work was also supported by the Light Microscopy Facility, a Core Facility of the CMCB Technology Platform at TU Dresden. We thank D. Bertheloot, D. De Nardo, Y. Herzig, B. G. Monks, L. Secchim Ribeiro, G. Engels and P. Leka (all from the Institute of Innate Immunity in Bonn) for providing reagents and expertise, as well as N. Zimmermann from the Department of Dermatology, Medical Faculty Carl Gustav Carus, Technische Universität Dresden for outstanding technical assistance. This work was funded in part by the European Union’s Horizon 2020 research and innovation program under grant agreement no. 848146 (To_Aition) (to E.L.), by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy–EXC2151–390873048 (to E.L.), by the DFG SFB1454-432325352 (to E.L.), SFB1402–414786233 (to E.L. and F.I.S.), TRR237–369799452 (to E.L. and C. Günther) and GRK2168–272482170 (to E.L.). This work was further supported by the Helmholtz-Gemeinschaft, Zukunftsthema ‘Immunology and Inflammation’ (ZT-0027, to E.L.) and the National Research Foundation-Prime Minister’s office, Republic of Singapore: NRF Fellowship 2018 (NRF-NRFF11-2019-0006) (to F.L.Z.) as well as the Nanyang Technological University: NAP2018 (to F.L.Z.) and an A*STAR Career Development Award (to K.S.R.).

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Author notes

  1. These authors contributed equally: Matilde B. Vasconcelos, Kim S. Robinson, Matthew S. J. Mangan.

Authors and Affiliations

  1. Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany

    Tomasz Próchnicki, Matilde B. Vasconcelos, Matthew S. J. Mangan, Dennis De Graaf, Kateryna Shkarina, Marta Lovotti, Lena Standke, Romina Kaiser, Rainer Stahl, Fraser G. Duthie, Maximilian Rothe, Kateryna Antonova, Lea-Marie Jenster, Dagmar Wachten, Florian I. Schmidt & Eicke Latz

  2. A*STAR Skin Research Labs (A*SRL), Clinical Sciences Building, Singapore, Singapore

    Kim S. Robinson & Zhi Heng Lau

  3. Skin Research Institute of Singapore (SRIS), Clinical Sciences Building, Singapore, Singapore

    Kim S. Robinson & Zhi Heng Lau

  4. Department of Dermatology, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany

    Sarah Rösing & Claudia Günther

  5. Institute of Nutritional Medicine, Department of Immunology, University of Hohenheim, Stuttgart, Stuttgart, Germany

    Nora Mirza, Clarissa Gottschild & Thomas A. Kufer

  6. Skin Research Institute of Singapore (SRIS), Immunos, Singapore, Singapore

    Franklin L. Zhong

  7. Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

    Franklin L. Zhong

  8. German Center of Neurodegenerative Diseases (DZNE), Bonn, Germany

    Eicke Latz

  9. Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA

    Eicke Latz

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  1. Tomasz Próchnicki
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Contributions

T.P., M.S.J.M., D.W. and E.L. conceived the project. T.P. and E.L. wrote the original draft. T.P., M.B.V., M.S.J.M., K.S.R., S.R., D.W., T.A.K., F.L.Z. and E.L. reviewed and edited the paper. T.P., M.B.V., M.S.J.M., K.S.R., S.R., F.G.D., L.S., R.K., M.R., T.A.K., F.I.S., D.D.G., M.L., R.S., K.A., N.M. and L.-M.J. devised the methodology. T.P., M.B.V., D.D.G., K.S.R., S.R., C. Gottschild, K.S., F.L.Z. and E.L. carried out the investigations. T.P., M.B.V. and E.L. provided the visualization. Funding acquisition was by C. Günther, F.L.Z., F.I.S. and E.L.

Corresponding author

Correspondence to Eicke Latz.

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Competing interests

E.L. is cofounder and consultant of IFM Therapeutics and Odyssey Therapeutics as well as a cofounder and board member of Dioscure Therapeutics and a Stealth Biotech. The other authors declare no competing interests.

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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: N. Bernard, in collaboration with the Nature Immunology editorial team.

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Extended data

Extended Data Fig. 1 m-3M3FBS activates the inflammasome independent of NLRP3.

(a) IL-1β secretion from WT iMacs, LPS-primed (200 ng/ml) and stimulated with nigericin (10 μM), poly-(dA:dT) (2 μg/ml) or m-3M3FBS (80 μM) (n = 3). (b) ASC specks per nuclei in iMacs expressing NLRP3 and ASCmCerulean, stimulated as in (a) (n = 3). (c) IL-1β secretion from WT iMacs, LPS-primed (200 ng/ml) and stimulated with nigericin (10 μM), poly-(dA:dT) (2 μg/ml) or m-3M3FBS (85 μM), in the presence of increasing doses of KCl (0, 5, 10, 25, 50, 75, 100, 125 mM) (n = 3). (d) ASC specks per nuclei in iMacs expressing NLRP3 and ASCmCerulean, stimulated as in (c), in the presence of increasing doses of KCl (0, 5, 10, 25, 50, 75, 100, 125 mM) (n = 3). (e) ASC specks per nuclei in WT iMacs expressing ASCmCerulean, with or without LPS priming (200 ng/ml) (41, 42), stimulated as in (c). While nigericin-induced ASC speck formation depends on priming, m-3M3FBS and poly-(dA:dT) trigger ASC speck formation in both LPS-primed and -unprimed cells (n = 3). (f) IL-1β secretion from WT iMacs, LPS-primed (200 ng/ml) and stimulated as in (c), in the presence or absence of the NLRP3 inhibitor CRID3 (5 μM) (43-45) (n = 3). (g) ASC specks per nuclei in iMacs expressing NLRP3 and ASCmCerulean, stimulated as in (c), in the presence or absence of the NLRP3 inhibitor CRID3 (5 μM) (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons test. **P = 0.0068, ****P < 0.0001, ns P = 0.5730-0.9999. (h) ASC speck formation in HEK cells stably expressing ASCTagBFP and transfected with an empty vector (EV) or human NLRC4, Pyrin or AIM2, stimulated with increasing doses of m-3M3FBS (40, 55, 70, or 85 μM); normalized to the unstimulated control (n = 3). (i) ASC specks per nuclei in HEK cells expressing human NLRP10 or mCitrine and ASCTagBFP, stimulated with nigericin (10 μM), poly-(dA:dT) (2 μg/ml) or m-3M3FBS (85 μM) (n = 4). Individual data points are means of technical triplicates (a, b, e, h, i) or duplicates (c, d, f, g); error bars represent SD.

Source data

Extended Data Fig. 2 NLRP10 does not colocalize with the ER, endolysosomal or Golgi compartments.

(a–c) Representative confocal micrographs of HEK cells stably expressing NLRP10mCherry and transfected with an endoplasmic reticulum marker (DDOSTmCitrine) (a), an endolysosomal compartment marker (LAMP1mCitrine) (b), or a Golgi apparatus marker (TGOLN2mCitrine) (c), stimulated with m-3M3FBS (85 μM) or nigericin (10 μM) (n = 4).

Extended Data Fig. 3 NLRP10 colocalizes with mitochondria and nucleates ASC specks upon thapsigargin and SMBA1 treatments.

(a) Representative confocal micrographs of HEK cells stably expressing NLRP10mCherry and transfected with TOMM20mCitrine mitochondrial marker, stimulated with thapsigargin (20 μM) or SMBA1 (50 μM) (n = 4). (b) ASC specks per nuclei in HEK cells expressing NLRP10 and ASCTagBFP, stimulated with m-3M3FBS (85 μM), thapsigargin (20 μM) or increasing doses of SMBA1 (5, 10, or 50 μM) (n = 3). Individual data points (where applicable) are means of technical duplicates (b); all error bars represent SD.

Source data

Extended Data Fig. 4 Upon mitochondrial permeabilization, AIM2 but not NLRP10 activation requires mtDNA.

(a) IL-1β secretion from WT iMacs, LPS-primed (200 ng/ml) and stimulated with thapsigargin (20 μM) (n = 3) or SMBA1 (50μM) (n = 4), in the presence or absence of the NLRP3 inhibitor CRID3 (5 μM) (43-45). P values were calculated by unpaired two-tailed Student’s t-test. ns P = 0.8492–0.8931. (b) IL-1β secretion from WT or AIM2-deficient bone marrow-derived macrophages, primed with LPS (200 ng/ml) and stimulated with poly-(dA:dT) dsDNA (2 μg/ml) or SMBA1 (50 μM) (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparison test. ****P < 0.0001. (c) ASC specks per nuclei in iMacs expressing NLRP3 and ASCmCerulean, stimulated with nigericin (10 μM), poly-(dA:dT) dsDNA (2 μg/ml), m-3M3FBS (85 μM), thapsigargin (20 μM) or SMBA1 (50 μM) (n = 4 for treatment with SMBA1 and 3 for other activators). (d, e) qPCR assessment of the whole-cell mtDNA content in WT iMacs (d) or HEK cells expressing NLRP10 and ASCTagBFP (e), cultured for 72–96 h with 2’,3’-dideoxycytidine (ddC) (80 μg/ml) (n = 3). (f) ASC specks per nuclei in HEK cells expressing NLRP10 and ASCTagBFP, cultured in the presence or absence of ddC (80 μg/ml), and stimulated with m-3M3FBS (85 μM) or thapsigargin (20 μM) (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons test. ns P = 0.1337–0.9909. Individual data points are values of technical monoplicates (d and e) or means of duplicates (a, c) or triplicates (b, f); all error bars represent SD.

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Extended Data Fig. 5 Differential sensitivity to cyclosporin A links mitochondrial damage to NLRP10 and AIM2 activation.

(a, b) Representative micrographs from time-lapse microscopy of HEK cells expressing NLRP10, ASCTagBFP and mCherry targeted to the mitochondrial matrix (a) or iMacs expressing NLRP3, ASCmCerulean and mCitrine targeted to the mitochondrial matrix (b) (using cytochrome c oxidase subunit 8 mitochondrial targeting sequence). Cells were treated with m-3M3FBS (85 μM) or thapsigargin (20 μM), with or without cyclosporin A (CsA; 15 μM) pre-treatment (n = 4). Images of the same field at treatment onset (0 min) or indicated time points.

Extended Data Fig. 6 NLRP10 activation by SMBA1 depends on mitochondrial permeability transition.

(a, c) ASC specks per nuclei in HEK cells expressing NLRP10 and ASCTagBFP (a) or iMacs expressing NLRP3 and ASCmCerulean (c), treated with SMBA1 (50 μM), in the presence or absence of cyclosporin A (CsA; 10 μM) (n = 3). P values were calculated by unpaired two-tailed Student’s t-test. ***P = 0.0009, ****P < 0.0001. (b, d) Representative endpoint fluorescence micrographs of HEK cells expressing NLRP10 and ASCTagBFP (b) or iMacs expressing NLRP3 and ASCmCerulean (d) with stable expression of mCitrine targeted to the mitochondrial matrix (using mitochondrial targeting sequence from cytochrome c oxidase subunit 8), stimulated with SMBA1 (50 μM), in the presence or absence of CsA (10 μM). Note that in the presence but not in the absence of CsA pre-treatment, mCitrine retains the granular distribution pattern consistent with mitochondrial localization (n = 3). Individual data points (where applicable) are means of technical duplicates; all error bars represent SD.

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Extended Data Fig. 7 Debio025 and NIM811 inhibit thapsigargin- but not m-3M3FBS-induced NLRP10 activation.

(a, d) ASC specks per nuclei in HEK cells expressing NLRP10 and ASCTagBFP, stimulated with m-3M3FBS (85 μM) or thapsigargin (20 μM), in the presence or absence of Debio025 (10 μM) (a) or NIM811 (10 μM) (d) (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons test. *P = 0.0276, **P = 0.0038, ns P = 0.0778-0.0792. (b, e) ASC specks per nuclei in iMacs expressing NLRP3 and ASCmCerulean, stimulated with nigericin (10 μM), poly-(dA:dT) dsDNA (2 μg/ml), m-3M3FBS (85 μM) or thapsigargin (20 μM), in the presence or absence of Debio025 (10 μM) (b) or NIM811 (10 μM) (e) (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons test. ***P = 0.0002, ****P < 0.0001, ns P = 0.2813-0.9999. (c, f) IL-1β secretion from WT iMacs, primed with LPS (200 ng/ml) and stimulated as in (b, e), in the presence or absence of Debio025 (10 μM) (c) or NIM811 (10 μM) (f) (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons test. **P = 0.0027, ****P < 0.0001, ns P = 0.1174-0.9996. Individual data points are means of technical duplicates; all error bars represent SD.

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Extended Data Fig. 8 NLRP10 activation upon m-3M3FBS treatment is independent of calcium fluxes.

(a, b) ASC specks per nuclei in HEK cells expressing NLRP10 and ASCTagBFP, with or without m-3M3FBS (85 μM) treatment, in the presence or absence of BAPTA-AM (50 μM) (a) or 2-APB (50 μM) (b) in Ca2+-free medium (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons test. **P = 0.0019, ns P = 0.3923. (c) Representative micrographs from time-lapse microscopy of HEK cells expressing NLRP10, ASCTagBFP and mCherry targeted to the mitochondrial matrix (using cytochrome c oxidase subunit 8 mitochondrial targeting sequence), treated with ionomycin (10 μM) in the presence of CaCl2 (1 mM). Images of the same field at treatment onset (0 min) or indicated time point (30 min) (n = 4). (d) Quantification of mitochondrial fluorescence signal granularity and ASC specks per imaging field (n = 4). (e) ASC specks per nuclei in HEK cells expressing NLRP10 and ASCTagBFP, stimulated with m-3M3FBS (85 μM) or with increasing doses of ionomycin (5, 10, 15 μM) in the presence of CaCl2 (1 mM) (n = 3). (f) ASC specks per nuclei in HEK cells expressing NLRP10 and ASCTagBFP, with or without m-3M3FBS (85 μM) treatment, in the presence or absence of ionomycin (15 μM) in Ca2+-free medium (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons test. *P = 0.0155. Individual data points are means of technical duplicates (a, b, e, and f) or means from 4 independent experiments (d); all error bars represent SD.

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Extended Data Fig. 9 Impact of emricasan on IL-1β secretion and ASC speck formation during mitochondrial rupture.

(a) IL-1β secretion from WT iMacs, primed with LPS (200 ng/ml) and stimulated with nigericin (10 μM), poly-(dA:dT) dsDNA (2 μg/ml), m-3M3FBS (85 μM) or thapsigargin (20 μM), in the presence or absence of emricasan (5 μM) (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons test. ****P < 0.0001. (b) ASC specks per nuclei in HEK cells expressing NLRP10 and ASCTagBFP, stimulated with m-3M3FBS (85 μM) or thapsigargin (20 μM), in the presence or absence of emricasan (5 μM) (n = 3). (c) ASC specks per nuclei in iMacs expressing NLRP3 and ASCmCerulean, stimulated as in (a), in the presence or absence of emricasan (5 μM) (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons test. ns P = 0.4112-0.9999. Individual data points are means of technical duplicates; all error bars represent SD.

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Extended Data Fig. 10 NLRP10 expression in primary and immortalized keratinocytes.

(a) qPCR analysis of NLRP10 mRNA expression in primary human keratinocytes, undifferentiated or differentiated for 6 days in medium containing CaCl2 (0.1–1 mM) (n = 2). (b) Representative western blot analysis of the differentiation marker involucrin and NLRP10 in undifferentiated or differentiated immortalized N/TERT human keratinocytes (n = 3). (c) Representative western blot analysis of NLRP10 in WT, Cas9 control or NLRP10-deficient (NLRP10sg2, NLRP10sg3) immortalized N/TERT human keratinocytes (n = 3). (d) Representative widefield fluorescence microscopy analysis of caspase-1 CARD domain aggregate formation in NLRP10-overexpressing or NLRP10-deficient immortalized N/TERT human keratinocytes, expressing Casp1-CARDeGFP, with or without m-3M3FBS (85 μM) treatment (n = 1). (e) Representative immunofluorescence staining and widefield fluorescence microscopy of NLRP10-overexpressing or NLRP10-deficient immortalized N/TERT human keratinocytes (n = 1). (f, g) Representative immunofluorescence staining and confocal microscopy of primary normal human keratinocytes, with or without m-3M3FBS (85 μM) treatment. Shown as maximum intensity projection of the z-stack, from an orthogonal (f) or top (g) view (n = 3). (h) ASC specks per nuclei in HEK cells expressing NLRP10 (either WT or the R243W variant) and ASCTagBFP, stimulated with m-3M3FBS (85 μM) or thapsigargin (20 μM) (n = 3). P values were calculated by 2-way ANOVA with Šídák’s multiple comparisons test. ***P = 0.0002-0.0005. Individual data points are means of technical duplicates (a, h); all error bars represent SD.

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Próchnicki, T., Vasconcelos, M.B., Robinson, K.S. et al. Mitochondrial damage activates the NLRP10 inflammasome. Nat Immunol 24, 595–603 (2023). https://doi.org/10.1038/s41590-023-01451-y

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